The formation and disassociation of molecular complexes are crucial to regulatory processes in living organisms. In particular, signaling pathways between the surface and nucleus of cells involve the formation of many molecular complexes in which multiple proteins are assembled to directly or indirectly induce molecular events, such as phosphorylation or dephosphorylation, which are steps in the signaling process, Gomperts et al, Signal Transduction (Academic Press, New York, 2002). Such pathways and their components have been the subject of intense investigation because of the role aberrant pathway behavior plays in many disease conditions, especially cancer, e.g. McCormick, Trends in Cell Biology, 9: 53-56 (1999); Blume-Jensen and Hunter, Nature, 411: 355-365 (2001); Nicholson et al, Cellular Signalling, 14: 381-395 (2002); and the like. It has been observed that many cancers are associated with an accumulation of mutations or other genetic alterations that affect components of signaling pathways, e.g. by over expression, particularly those pathways involved with cell proliferation, cell motility, differentiation, and cell death, e.g. Blume-Jensen and Hunter (cited above). Unfortunately, such signaling pathways have been difficult to study not only because of their complexity and interconnectedness, but also because of the disruptive procedures required for analysis of intracellular complexes, e.g. Weng et al, Science, 284: 92-96 (1999); Machida et al, Molecular & Cellular Proteomics, 2.4: 215-233 (2003); Price et al, Methods in Molecular Biology, 218: 255-267 (2003). In particular, the study of phosphorylation states of signaling proteins has been difficult because during sample preparation the phosphorylation state is rapidly altered by the actions of enzymes, such as phosphatases.
A wide variety of techniques have been used to study cellular protein-protein interactions and complexes, including immunoprecipitation, chemical cross-linking, bioluminescence resonance energy transfer (BRET), fluorescence resonance energy transfer (FRET), and the like, e.g. Price et al (cited above); Sorkin et al, Curr. Biol., 10: 1395-1398 (2000); McVey et al, J. Biol. Chem., 17: 14092-14099 (2001); Salim et al, J. Biol. Chem., 277: 15482-15485 (2002); Angers et al, Proc. Natl. Acad. Sci., 97: 3684-3689 (2000). Unfortunately, such techniques are difficult to apply, and generally lack sufficient sensitivity to provide an accurate picture of the state of a signaling pathway.
In view of the above, the availability of a convenient, sensitive, and cost effective technique for simultaneously detecting or measuring one or more molecular complexes, particularly those in signaling pathways, would advance many fields where such measurements are becoming increasingly important, including life science research, medical research and diagnostics, drug discovery, and the like.